富含二硫键的膜蛋白p185胞外区在大肠杆菌中的可溶性表达和性质鉴定

Her2/c-erbB-2基因（其产物为膜蛋白p185）是表皮生长因子受体（EGFR）基因家族的一员，在约30％的乳腺癌中发现了其过量表达。为了鉴定抗p185单克隆抗体的抗原表位并进一步研究它们的相互作用，采用PCR的方法从含Her2/c_erbB_2基因的pBabe/erbB_2质粒中扩增了p185胞外区的富含二硫键的第一、二结构域和第四个结构域。产物克隆到pGEX/4T-1载体后，转化大肠杆菌Origami B（DE3）pLysS菌株，用低浓度IPTG进行低温过夜诱导后将菌体压力破碎，SDS-PAGE检测表达上清，得到了可溶性表达的融合有GST的目的蛋白。经ELISA、Western blot等方法鉴定，可溶性表达产物具有完全的抗体结合活性，且当用凝血酶把GST切掉后该活性仍然保留。P185胞外区融合蛋白的成功表达将为二硫键富含类蛋白的表达提供参考；并为将来具有肿瘤细胞生长抑制活性的抗p185单克隆抗体的抗原表位鉴定，以及为EGFR家族受体的结构和功能关系的研究打下基础。

Soluble Expression and Characterization of Disulfide Bond-rich Subdomains of Membrane Protein p185 in Escherichia coli

Transmembrane protein p185 (the product of Her2/c-erbB-2 gene) is a member of the epidermal growth factor receptor (EGFR) family. Its overexpression was found in about 30% of breast cancer. It is essential to obtain soluble extracellular domain (ECD) of p185, especially disulfide bond rich domains, for identifying the epitopes of anti-p185 antibodies and researching the interrelationship between the antigen and antibody. The disulfide bond rich domain I-II and domain IV of p185 ECD were amplified from plasmid pBabe/erbB-2 by PCR respectively. These two fragments were inserted into pGEX/4T-1 vector, transfected into E. coli Origami B (DE3) pLysS and expressed inductively by low concentration of IPTG and low temperature overnight. After the pressure lysis of cells, the supernatants were analyzed by SDS-PAGE and the result demonstrated that this GST-fusion protein was expressed solubly in the amount of 10-15 mg/L. By the ELISA, Western blot and other immunological assays, the fusion proteins and their GST cut-off derivates both showed binding activities with several anti-p185 antibodies respectively. These results indicated that it was a feasible and effectual method to express disulfide bond rich domain I-II and domain IV of p185 ECD and this method may also be used to express other disulfide bond rich proteins.